The immobilized enzyme technology is used widely in such fields as food production, production of fine chemicals and solution of environmental problems by means of bioreactors. Said technology is also being used in the area of analytical chemistry, where the high substrate specificity, high reaction rate and high sensitivity characteristics of enzymes are utilized. Analytical devices utilizing immobilized enzymes are generally known as biosensors and now are being studied widely.
Biosensors can be classified, by the detection technique employed therein, into electrode, field effect transistor, photodetector, thermal detector and other types. Among these, the electrode-type biosensors are simple in construction and can be provided with added specificity by selection of the electrode as well as the enzyme species used. Furthermore, unlike field effect transistor-type biosensors, they are scarcely influenced by pH and, unlike photodetection-type biosensors, they are virtually indifferent to the color or turbidity of samples. Accordingly, various apparatus or devices based on biosensors of the electrode type in particular have been developed.
Biosensors using electrodes are further classifiable into two major types, namely amperometric sensors in which an oxygen electrode, a hydrogen peroxide electrode or the like is used and potentiometric sensors which employ an ion selective electrode such as a pH electrode or an ammonia electrode. Amperometric sensors are used more frequently since they give a linear relationship between substance concentration and output electric current value, which facilitates data processing and make it possible to construct high-accuracy and high-sensitivity apparatus with a simple structure.
Since the measurement technique using biosensors is advantageous not only in accuracy and sensitivity as mentioned above but also in the ease and simplicity of operation and data processing, the range of application of this technique is expanding.
However, most of the biosensors available today employ an oxidase and measure the concentration of oxygen which decreases or the concentration of hydrogen peroxide which increases. In other words, with these biosensors, the targets of measurement are limited to substances of the oxidase employed. Therefore, assay systems employing a variety of other enzymes such as hydrolase, isomerase, dehydrogenase, etc., each in immobilized form, together with an immobilized oxidase are also in use. The use of these immobilized enzymes is advantageous in that the range of targets of assay is extended, the measurement sensitivity can be improved and the rate of reaction can be increased, for instance.
Among such assay systems, the assay system for lactic acid and pyruvic acid is attracting particular attention because of its importance in, inter alia, fermentation control and clinical diagnosis. A typical example can be found in alcohol fermentation. In alcohol fermentation, it is important to know both the concentration of lactic acid and that of pyruvic acid as indices of whether the metabolic system of the yeast is working normally or not. This is particularly important in the process for brewing sake.
As known examples of the assay system, the following may be mentioned.
(1) The method comprising immobilizing L-lactate oxidase on a carrier and assaying lactic acid based on the quantity of hydrogen peroxide formed.
(2) The method comprising immobilizing L-lactate dehydrogenase on a carrier, reducing pyruvic acid in the presence of NADH and assaying pyruvic acid based on the decrease in absorbance at 340 nm.
(3) The method comprising contacting pyruvic acid with pyruvate oxidase in the presence of FAD.sup.+, TPP (thiamine pyrophosphonate) and Mg.sup.2+ to form acetylphosphate and assaying pyruvic acid based on the change in quantity of hydrogen peroxide or oxygen.
(4) The method comprising co-immobilizing L-lactate oxidase and L-lactate dehydrogenase, bringing a substrate into contact with both immobilized enzymes simultaneously to thereby reduce the substrate which has been oxidized by oxidase-catalyzed reaction by means of the dehydrogenase and re-oxidize the thus-reduced substrate by oxidase-catalyzed reaction in repetition and assaying either pyruvic acid or lactic acid or both together, with high sensitivity. This assay method utilizes the principle of enzyme cycling, i.e., the phenomenon that a larger number of molecules of oxygen than molecules of the substrate are converted to hydrogen peroxide (Japanese Examined Patent Publication No. 3-65492).
The methods (1) and (2) mentioned above can assay only either lactic acid or pyruvic acid. In cases where it is necessary to assay both, lactic acid and pyruvic acid must be assayed separately. This is because, by the L-lactate oxidase lactic acid is oxidised to pyruvic acid in the method (1), hydrogen peroxide which can be measured with high sensitivity at the electrode is formed. And in the method (2), by the L-lactate dehydrogenase pyruvic acid is reduced, NADH, which is to be measured spectrophotemetrically, is decreased. Therefore if one tries to assay both of them simultaneously he cannot but fail because of the impossibility of precisely assaying both owing to the above difference in detection method.
The method (3) mentioned above has drawbacks that two coenzymes are required and pyruvate oxidase is not stable.
The disadvantage of the above cycling system (4) employing an enzyme combination is that once the enzymatic reaction has started, the oxidized form and the reduced form of the substrate for the dehydrogenase are both involved in the reaction. More specifically, when L-lactate dehydrogenase and L-lactate oxidase are immobilized and cycling is performed, pyruvic acid in the sample is first converted to L-lactic acid and at the same time L-lactic acid is converted to pyruvic acid by the oxidase-catalyzed reaction, if NADH (reduced-form nicotinamide adenine dinucleotide) coexists in the system. If NADH is present in a sufficient amount and the amount of dissolved oxygen is also sufficient, that cycle is repeated and consequently the hydrogen peroxide formed upon oxidation of lactic acid is accumulated. Therefore, when the decrease in the concentration of oxygen or the increase in the concentration of hydrogen peroxide is detected electrochemically, an improved sensitivity can be obtained as compared with the case where no cycling is performed. However, if the sample contains L-lactic acid from the beginning, this cannot be distinguished from pyruvic acid. In other words, the quantity of pyruvic acid cannot be determined exactly.
The prior art assay methods using immobilized enzymes thus encounter the problems mentioned above. No effective apparatus have been disclosed as yet for reaction systems in which a dehydrogenase is utilized. This is particularly because the dehydrogenase-catalyzed oxidation of substrates in the presence of NAD.sup.+ can hardly proceed, hence it is a general practice to use an oxidase, and because the dehydrogenase-catalyzed oxidation of substrates produces NADH, which is not suited for high-sensitivity sensors.
The present invention relates to an assay apparatus and assay method in which a dehydrogenase in immobilized form and an oxidase in immobilized form are utilized. The object of the invention is to provide a multifunctional assay apparatus and assay method by which two components, namely an oxidized-form substrate (or a reduced-form coenzyme), and a reduced-form substrate of a dehydrogenase, can be simultaneously assayed.